Dermal Papilla Cell Proliferation of Phytochemicals Isolated from Chestnut Shells (Castanea crenata)

Castanea crenata (Fagaceae) is a species of chestnut tree that is endemic to the Republic of Korea and Japan. While its kernels are consumed, chestnut by-products such as shells and burs, which account for 10–15% of the total weight, are discarded as waste. Phytochemical and biological studies have been carried out to eliminate this waste and develop high-value products from its by-products. In this study, five new compounds (1–2, 6–8) along with seven known compounds were isolated from the shell of C. crenata. This is the first study to report diterpenes from the shell of C. crenata. Comprehensive spectroscopic data including 1D, 2D NMR, and CD spectroscopy were used to determine the compound structures. All isolated compounds were examined for their ability to stimulate dermal papilla cell proliferation using a CCK-8 assay. In particular, 6β,7β,16α,17-Tetrahydroxy-ent-kauranoic acid, isopentyl-α-L-arabinofuranosyl-(1→6)-β-D-glucopyranoside, and ellagic acid exhibited the most potent proliferation activity of all.


Introduction
Castanea crenata, commonly referred to as Republic of Korean or Japanese chestnut, is an indigenous species of chestnut tree found in the Republic of Korea and Japan. The Republic of Korea ranks as the world's second-highest producer of chestnuts, but 30% of them are processed manually as peeled chestnuts [1]. The process of peeling chestnuts generates two waste products: the burs and the shells. In Asia, the chestnut shell is used for its medicinal properties, including alleviating diarrhea and improving digestion [2]. Despite being a rich source of bioactive compounds, chestnut shells are considered solid waste and often burned as fuel in factories, leading to environmental problems caused by the release of poisonous substances such as CO, NOx, and polychlorodibenzodioxins [3]. Our previous studies have identified cycloartane-type triterpenoids and flavonoid glycosides in C. crenata burs, which have contributed to the discovery of natural antiviral sources [4]. Likewise, chestnut shells contain various health-beneficial compounds such as polyphenols and flavonoids, which have been shown to have antioxidant, anticancer, anti-inflammation, antibacterial, diabetes control, and weight loss effects [1,3,5].
Hair loss is a result of various factors, including aging, hormonal imbalances, nutrient deficiencies, and psychological stress. The hair follicle dermal papilla cells (DPCs) found at the base of the hair follicle are being employed in an in vitro screening model for hair growth, as they play a crucial role in hair follicular morphogenesis and postnatal hair growth cycles [6]. In our preliminary study, we discovered that (3R)-5 -methoxyvestiol and (6aR,11aR)-3,8-dihydroxy-9-methoxypterocarpan significantly impacted the proliferation Plants 2023, 12, 1018 2 of 10 of immortalized DPCs [7,8]. Additionally, chestnut shell extracts demonstrated minimal internal damage with excellent color intensity and a smooth hair surface morphology [5]. Thus, combining phytochemical exploration with biological activity investigation is crucial in discovering natural hair growth compounds from agricultural by-products. In this study, five previously undescribed compounds (1-2, 6-8) along with seven known compounds were purified from C. crenata shells. Despite extensive investigation into the phenolic compounds in chestnut shells [2,9], no diterpenoid derivatives have been identified to date.

Dermal Papilla Cell Proliferation
Phytochemicals have been explored as potential hair growth stimulants due to their low toxicity, accessibility, affordability, and diverse modes of biochemical action [6][7][8].
Despite substantial studies on natural products for hair growth, the findings of compoundbased research still require a more in-depth understanding, since the majority of these studies have used natural product extracts. In this investigation, the effects of all isolated compounds on dermal papilla cell proliferation were evaluated using a CCK-8 assay and the findings are summarized in Table 3. Table 3. Dermal papilla cell proliferation effect of isolated compounds.
Vanillic acid and hydroxytyrosol, simple phenolic acids found in wheat bran and olive oil, enhance DPCs' proliferation. Vanillic acid stimulates anagen and reduces hair loss in DPCs, whereas hydroxytyrosol increases the release of hair growth factors during the anti-inflammatory process [26,27]. Research into the effects of phenolic glycosides on hair growth has not been extensively conducted. Nevertheless, all the phenolic glycosides isolated from the chestnut shells, except 2-phenylethyl β-rutinoside (10), in the present study displayed significant activity, with proliferation ranging from 117 to 138.
In accordance with the results from the present investigation, previous research has demonstrated that ellagic acid (11) has a significant effect in stimulating hair growth by extending the follicular size and prolonging the growing phase [28]. Additionally, ellagic acid protected irradiated hair follicle dermal papilla cells from UVA-induced damage by exhibiting an ROS scavenging capacity and modulating antioxidant gene expression [29]. These findings support the potential use of chestnut by-products as a source of high-value compounds and the possibility that diterpenoids and phenolic glycosides derived from chestnut shells might be promising candidates for hair-growth-enhancing agents. Further research is being undertaken to understand the mechanism behind their proliferation effects.

Plant Material
Castanea crenata shells from Gongju-si, Chungcheongnam-do, Republic of Korea, were obtained from the Kyung-dong herbal market in Seoul, Republic of Korea, in 2019. The Okkwang cultivar, widely cultivated in the Republic of Korea, was used for this study [2]. A voucher specimen (CC201901) was deposited in the Herbarium of the College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, Republic of Korea.  Table 1.

Cell Culture and Cell Proliferation Assay
Immortalized dermal papilla cells (iDPCs) were obtained from Dr. Sung's Lab at Kyungpook National University and cultured in DMEM containing 10% FBS (Gibco, Rockville, MD, USA) and 1% penicillin/streptomycin (Gibco, CA, USA) at 37 • C in a humidified atmosphere of 95% air/5% CO 2 . A total of 1000 iDPCs cells per well were plated in 96-well microplates and treated with 30 µM of isolated compounds in DMSO as well as minoxidil, the positive control, for 24 h. The cell proliferation was measured using the CCK-8 solution (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). The absorbance was measured at 450 nm using a microplate reader (Tecan, AG, Switzerland). The percentage of cell proliferation was calculated by setting the control group without compound (blank) as 100%.

Statistical Analysis
The statistical software package GraphPad Prism (ver. 5, GraphPad, San Diego, CA, USA) was used to examine the data. All data are presented as means ± standard error of the mean (SEM). The statistical significance of the differences between the compounds and the control group (blank) was determined using a paired t-test. p-values were considered statistically significant when <0.05 (*), <0.01 (**), and <0.001 (***).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.